Method to improve virus filtration capacity

ABSTRACT

The present invention relates to the field of protein purification. In particular, the invention concerns methods for increasing the filtration capacity of virus filters, by combined use of endotoxin removal and cation-exchange media in the prefiltration process.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/806,171, filed on Aug. 6, 2010, which claims priority under 35 U.S.C.Section 119(e) and the benefit of U.S. Provisional Application No.61/231,811, filed Aug. 6, 2009, the entire disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention is from the field of protein purification. Inparticular, the invention concerns methods for increasing the filtrationcapacity of virus filters, by combined use of endotoxin removal andcation-exchange media in the prefiltration process.

Description of the Related Art

Mammalian cell lines have become the primary choice for production ofrecombinant protein therapeutics due to their capacity for properprotein folding and post translational modification such asglycosylation (Chu and Robinson Current Opinion in Biotechnology12:180-187, 2001). However, these cell lines are also known to containretrovirus like particles (Lieber et al. Science 182:56-59, 1973;Lubiniecki et al. Dev Biol Stand 70:187-191, 1989) and possess the riskfor potential adventitious virus contamination (Garnick, Dev Biol Stand.Basel: Karger 93:21-29, 1998). While the biopharmaceutical industryproducing recombinant protein drugs has a good safety record, there havebeen past incidences of viral infection by blood and blood productsderived from plasma (Brown, Dev. Biol. Stand. 81, 1993; Thomas, Lancet343:1583-1584, 1994). To mitigate the risk of viral contamination duringrecombinant protein production, downstream purification processes aredesigned to include process steps that remove endogenous andadventitious viruses. Adequate virus clearance is obtained by acombination of several process steps that provide either virusinactivation or virus removal from the process feed stream. While viralinactivation is achieved using techniques such as incubation at low pH,heat treatment, and detergents, virus removal is typically performedusing chromatography and filtration (Curtis et al., Biotechnology andBioengineering 84(2):179-186, 2003).

Unlike chromatography media, which removes viruses based onphysicochemical properties such as net charge, virus filtration removesviruses by size exclusion and is therefore considered a more robusttechnique. So far usage of virus filtration during downstreampurification of biotherapeutics derived from mammalian cell cultures hasbeen limited to removal of retroviruses (80-100 nm diameter) due to lackof high throughput membranes with nominal pore size less than 60 nm.

Recent advances in membrane technology have enabled manufacturing ofhigh throughput membranes with nominal pore size of 20 nm. These virusfilters are retentive to parvoviruses (18-26 nm diameter) and allowpassage of proteins that are as large as 160 kD (˜8 nm), e.g.,monoclonal antibodies (mAbs).

The high selectivity and high throughput with parvovirus filters isachieved by casting a thin retentive membrane layer on a microporoussubstrate. The thin retentive layer while allows very fine separation ofproteins and viruses, it is also susceptible to fouling by impurities inthe process feedstream resulting in lower filter capacity and flux. Thefouling of the virus filters has been attributed to contaminants such asprotein aggregates and denatured protein. Bohonak and Zydney (Bohonakand Zydney, Journal of Membrane Science 254(1-2):71-79, 2005) showedthat loss in filter capacity could be due to cake formation or poreblockage. Other recent reports (Bolton et al., Biotechnol. Appl.Biochem. 43:55-63, 2006; Levy et al., Filtration in theBiopharamaceutical Industry. (Meltzer, T. H. and Jornitz, M. W., eds.)pp. 619-646, Marcel Dekker, New York, 1998) have attributed the likelycause of filter fouling to the adsorption of impurities to the porewalls. Several publications (Bolton et al., Biotechnology and AppliedBiochemistry 42:133-142, 2005; Hirasaki et al., Polymer Journal26(11):1244-1256, 1994; Omar and Kempf, Transfusion 42(8):1005-1010,2002) have also demonstrated that reduction in filter capacity orplugging of pores can decrease viral retention by few orders ofmagnitude, affecting the robustness of the unit operation.

A lot of recent research has thus focused on identification ofpre-filters for removing the foulants from the process feedstream tominimize virus filter fouling and ensuring high capacity, highthroughput and robust viral retention. Bolton et al. (Bolton et al.2006) performed a thorough study that involved testing of severalmembranes as prefilters and demonstrated that it was possible toincrease capacity of normal flow parvovirus (NFP) membranes by almost anorder of magnitude by using VIRESOLVE™ depth filter as a prefilter.Brown et al. (Brown et al. 2008, Use of Charged Membranes to IdentifySoluble Protein Foulants in order to Facilitate Parvovirus Filtration.IBC's 20^(th) Antibody Development and Production, San Diego, Calif.)evaluated a strong cation exchange membrane adsorber as a prefilter toparvovirus retentive filter and showed that the capacity of virus filtercould be increased by several fold for eleven different mAb streams. Theauthors hypothesized that the cation exchange membrane adsorber removedlarge molecular weight (˜600-1500 kD) protein aggregates from thefeedstream by competitive adsorption, preventing the virus filter fromplugging. U.S. Pat. No. 7,118,675 (Siwak et al.) describes a processthat utilizes a charge-modified membrane to remove protein aggregatesfrom a protein solution to prevent fouling of virus filter.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the experimentalfinding that fouling of parvovirus filters could be due to impuritiesother than those mentioned in the literature and more comprehensiveprefiltration solutions are required to improve the virus filtrationcapacity. Accordingly, the present invention provides a novelprefiltration solution that performs significantly better than the bestprefiltration approach mentioned in the literature (cation-exchangemembrane adsorbers).

In one aspect, the invention concerns a method of improving thefiltration capacity of a virus filter during protein purification,comprising subjecting a composition comprising a protein to be purifiedto a cation exchange step and an endotoxin removal step, in eitherorder, prior to passing through said virus filter.

In one embodiment, the pore size of the virus filter is between about 15and about 100 nm in diameter.

In another embodiment, the pore size of the virus filter is betweenabout 15 and about 30 nm in diameter.

In yet another embodiment, the pore size of the virus filter is about 20nm.

In a further embodiment, the virus to be removed is a parvovirus.

In a still further embodiment, the diameter of the parvovirus is betweenabout 18 and about 26 nm.

In a different embodiment, the protein is an antibody or an antibodyfragment, such as an antibody produced by recombinant DNA techniques, ora fragment thereof.

In another embodiment, the antibody is a therapeutic antibody.

In yet another embodiment, the recombinant antibody or antibody fragmentis produced in a mammalian host cell, such as, for example, a ChineseHamster Ovary (CHO) cell.

In a further embodiment, the composition comprising the protein to bepurified is first subjected to a cation exchange step followed by anendotoxin removal step, prior to virus filtration.

In a still further embodiment, the composition comprising the protein tobe purified is first subjected to an endotoxin removal step followed bya cation exchange step, prior to virus filtration.

In another embodiment, the composition comprising the protein to bepurified is subjected to a cation exchange step and endotoxin removalstep simultaneously, prior to virus filtration, by keeping the two mediatogether in a single module.

In yet another embodiment, the endotoxin removal step is directlyfollowed by virus filtration.

In a further embodiment, the cation exchange step is directly followedby virus filtration.

In a different embodiment, virus filtration is performed at a pH betweenabout 4 and about 10.

In another embodiment, the protein concentration in the composition tobe purified is about 1-40 g/L.

In yet another embodiment, the antibody to be purified is to one or moreantigens selected from the group consisting of HER1 (EGFR), HER2, HER3,HER4, VEGF, CD20, CD22, CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4, VCAM,IL-17A and/or F, IgE, DR5, CD40, Apo2L/TRAIL, EGFL7, NRP1, mitogenactivated protein kinase (MAPK), and Factor D.

In a further embodiment, the antibody is selected from the groupconsisting of anti-estrogen receptor antibody, anti-progesteronereceptor antibody, anti-p53 antibody, anti-cathepsin D antibody,anti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody,anti-CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB-2 antibody,anti-P-glycoprotein antibody, anti-CEA antibody, anti-retinoblastomaprotein antibody, anti-ras oncoprotein antibody, anti-Lewis X antibody,anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody,anti-CD9/p24 antibody, anti-CD10 antibody, anti-CD11c antibody,anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody,anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38antibody, anti-CD41 antibody, anti-LCA/CD45 antibody, anti-CD45ROantibody, anti-CD45RA antibody, anti-CD39 antibody, anti-CD100 antibody,anti-CD95/Fas antibody, anti-CD99 antibody, anti-CD106 antibody,anti-ubiquitin antibody, anti-CD71 antibody, anti-c-myc antibody,anti-cytokeratins antibody, anti-vimentins antibody, anti-HPV proteinsantibody, anti-kappa light chains antibody, anti-lambda light chainsantibody, anti-melanosomes antibody, anti-prostate specific antigenantibody, anti-S-100 antibody, anti-tau antigen antibody, anti-fibrinantibody, anti-keratins antibody and anti-Tn-antigen antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A schematic of the experimental setup used for virus filtrationstudies.

FIG. 2: Effect of sterile and depth filter on the capacity of ViresolvePro parvovirus retentive filter. Experiments were performed at pH 5.5and conductivity of 8.5 mS/cm. mAb concentration was approximately 13g/L.

FIGS. 3 (a) and (b): Effect of cation-exchange and endotoxin removalmembrane adsorbers as prefilters on the capacity of Viresolve Proparvovirus filter. The data in 3 (a) and 3 (b) were generated at pH 5.0and 6.5 respectively with MAb1.

FIGS. 4 (a) and (b): Effect of a novel prefiltration train containingboth cation-exchange and endotoxin removal membrane adsorbers on thecapacity of Viresolve Pro parvovirus retentive filter with MAb1. Thedata in 4 (a) and 4 (b) were generated at pH 5.0 and 6.5 respectively.

FIG. 5: Effect of a novel prefiltration train containing bothcation-exchange and endotoxin removal membrane adsorbers compared tocation-exchange pre-filtration media on the capacity of parvovirusretentive filter with MAb2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT I. Definitions

By “protein” is meant a sequence of amino acids for which the chainlength is sufficient to produce the higher levels of tertiary and/orquaternary structure. Thus, proteins are distinguished from “peptides”which are also amino acid-based molecules that do not have suchstructure. Typically, a protein for use herein will have a molecularweight of at least about 15-20 kD, preferably at least about 20 kD.

Examples of proteins encompassed within the definition herein includemammalian proteins, such as, e.g., CD4, integrins and their subunits,such as beta7, growth hormone, including human growth hormone and bovinegrowth hormone; growth hormone releasing factor; parathyroid hormone;thyroid stimulating hormone; lipoproteins; α-1-antitrypsin; insulinA-chain; insulin B-chain; proinsulin; follicle stimulating hormone;calcitonin; luteinizing hormone; glucagon; clotting factors such asfactor VIIIC, factor IX, tissue factor, and von Willebrands factor;anti-clotting factors such as Protein C; atrial natriuretic factor; lungsurfactant; a plasminogen activator, such as urokinase or tissue-typeplasminogen activator (t-PA, e.g., Activase®, TNKase®, Retevase®);bombazine; thrombin; tumor necrosis factor-α and -β; enkephalinase;RANTES (regulated on activation normally T-cell expressed and secreted);human macrophage inflammatory protein (MIP-1-α); serum albumin such ashuman serum albumin; mullerian-inhibiting substance; mousegonadotropin-associated peptide; DNase; inhibin; activin; vascularendothelial growth factor (VEGF); IgE, receptors for hormones or growthfactors; an integrin; protein A or D; rheumatoid factors; a neurotrophicfactor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3,-4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor suchas NGF-β; platelet-derived growth factor (PDGF); fibroblast growthfactor such as aFGF and bFGF; epidermal growth factor (EGF);transforming growth factor (TGF) such as TGF-α and TGF-β, includingTGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factor-Iand -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I); insulin-likegrowth factor binding proteins; other CD proteins such as CD3, CD8, CD19and CD20; erythropoietin (EPO); thrombopoietin (TPO); osteoinductivefactors; immunotoxins; a bone morphogenetic protein (BMP); an interferonsuch as interferon-α, -β, and -γ; colony stimulating factors (CSFs),e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10;superoxide dismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor (DAF); a viral antigen such as, for example, aportion of the AIDS envelope; transport proteins; homing receptors;addressins; regulatory proteins; integrins such as CD11a, CD11b, CD11c,CD18, an ICAM, VLA-4 and VCAM; a tumor associated antigen such as HER1(EGFR), HER2, HER3 or HER4 receptor; Apo2L/TRAIL, and fragments of anyof the above-listed polypeptides; as well as immunoadhesins andantibodies binding to; and biologically active fragments or variants ofany of the above-listed proteins.

Specifically included within the definition of “protein,” as definedherein, are therapeutic antibodies and immunoadhesins, including,without limitation, antibodies to one or more of the following antigens:HER1 (EGFR), HER2, HER3, HER4, VEGF, CD20, CD22, CD11a, CD11b, CD11c,CD18, an ICAM, VLA-4, VCAM. IL-17A and/or F, IgE, DR5, CD40,Apo2L/TRAIL, EGFL7, NRP1, mitogen activated protein kinase (MAPK), andFactor D, and fragments thereof.

Other exemplary antibodies include those selected from, and withoutlimitation, anti-estrogen receptor antibody, anti-progesterone receptorantibody, anti-p53 antibody, anti-cathepsin D antibody, anti-Bcl-2antibody, anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3antibody, anti-CA19-9 antibody, anti-c-erbB-2 antibody,anti-P-glycoprotein antibody, anti-CEA antibody, anti-retinoblastomaprotein antibody, anti-ras oncoprotein antibody, anti-Lewis X antibody,anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody,anti-CD9/p24 antibody, anti-CD10 antibody, anti-CD11c antibody,anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody,anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38antibody, anti-CD41 antibody, anti-LCA/CD45 antibody, anti-CD45ROantibody, anti-CD45RA antibody, anti-CD39 antibody, anti-CD100 antibody,anti-CD95/Fas antibody, anti-CD99 antibody, anti-CD106 antibody,anti-ubiquitin antibody, anti-CD71 antibody, anti-c-myc antibody,anti-cytokeratins antibody, anti-vimentins antibody, anti-HPV proteinsantibody, anti-kappa light chains antibody, anti-lambda light chainsantibody, anti-melanosomes antibody, anti-prostate specific antigenantibody, anti-S-100 antibody, anti-tau antigen antibody, anti-fibrinantibody, anti-keratins antibody and anti-Tn-antigen antibody.

An “isolated” protein, such as antibody, is one which has beenidentified and separated and/or recovered from a component of itsnatural environment. Contaminant components of its natural environmentare materials which would interfere with diagnostic or therapeutic usesfor the protein, such as antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes. In preferredembodiments, the protein, such as antibody, will be purified (1) togreater than 95% by weight as determined by the Lowry method, and mostpreferably more than 99% by weight, (2) to a degree sufficient to obtainat least 15 residues of N-terminal or internal amino acid sequence byuse of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGEunder reducing or nonreducing conditions using Coomassie blue or,preferably, silver stain.

The protein is preferably essentially pure and desirably essentiallyhomogeneous (i.e. free from contaminating proteins). “Essentially pure”protein means a composition comprising at least about 90% by weight ofthe protein, based on total weight of the composition, preferably atleast about 95% by weight.

“Essentially homogeneous” protein means a composition comprising atleast about 99% by weight of protein, based on total weight of thecomposition.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length antibodies whichhave an immunoglobulin Fc region), antibody compositions withpolyepitopic specificity, bispecific antibodies, diabodies, andsingle-chain molecules, as well as antibody fragments (e.g., Fab,F(ab′)₂, and Fv).

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. An IgM antibody consists of 5 of the basic heterotetramer unitalong with an additional polypeptide called a J chain, and contains 10antigen binding sites, while IgA antibodies comprise from 2-5 of thebasic 4-chain units which can polymerize to form polyvalent assemblagesin combination with the J chain. In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for μ and ε isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain at its other end. The V_(L) is aligned with the V_(H) and theC_(L) is aligned with the first constant domain of the heavy chain(C_(H)1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see e.g., Basic and Clinical Immunology, 8th Edition, DanielP. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange,Norwalk, Conn., 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (CH),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, havingheavy chains designated α, δ, ε, γ and μ, respectively. The γ and μclasses are further divided into subclasses on the basis of relativelyminor differences in the CH sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines the specificity of aparticular antibody for its particular antigen. However, the variabilityis not evenly distributed across the entire span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of about 15-30 amino acidresidues separated by shorter regions of extreme variability called“hypervariable regions” or sometimes “complementarity determiningregions” (CDRs) that are each approximately 9-12 amino acid residues inlength. The variable domains of native heavy and light chains eachcomprise four FRs, largely adopting a β-sheet configuration, connectedby three hypervariable regions, which form loops connecting, and in somecases forming part of, the β-sheet structure. The hypervariable regionsin each chain are held together in close proximity by the FRs and, withthe hypervariable regions from the other chain, contribute to theformation of the antigen binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Theconstant domains are not involved directly in binding an antibody to anantigen, but exhibit various effector functions, such as participationof the antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” (also known as “complementaritydetermining regions” or CDRs) when used herein refers to the amino acidresidues of an antibody which are (usually three or four short regionsof exteme sequence variability) within the V-region domain of animmunoglobulin which form the antigen-binding site and are the maindeterminants of antigen specificity. There are at least two methods foridentifying the CDR residues: (1) An approach based on cross-speciessequence variability (i.e., Kabat et al., Sequences of Proteins ofImmunological Interest (National Institute of Health, Bethesda, M S1991); and (2) An approach based on crystallographic studies ofantigen-antibody complexes (Chothia, C. et al., J. Mol. Biol. 196:901-917 (1987)). However, to the extent that two residue identificationtechniques define regions of overlapping, but not identical regions,they can be combined to define a hybrid CDR.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256: 495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is(are) identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855(1984)).

An “intact” antibody is one which comprises an antigen-binding site aswell as a CL and at least the heavy chain domains, C_(H)1, C_(H)2 andC_(H)3.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules and multispecific antibodies formed fromantibody fragments.

Papain digestion of antibodies produced two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (V_(H)), and the first constant domain of oneheavy chain (C_(H)1). Each Fab fragment is monovalent with respect toantigen binding, i.e., it has a single antigen-binding site. Pepsintreatment of an antibody yields a single large F(ab′)₂ fragment whichroughly corresponds to two disulfide linked Fab fragments havingdifferent antigen-binding activity and is still capable of cross-linkingantigen. Fab′ fragments differ from Fab fragments by having a fewadditional residues at the carboxy terminus of the C_(H)1 domainincluding one or more cysteines from the antibody hinge region. Fab′-SHis the designation herein for Fab′ in which the cysteine residue(s) ofthe constant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, the region which is alsorecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervarible loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of the sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10) residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,thereby resulting in a bivalent fragment, i.e., a fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described in greater detail in, for example, EP 404,097; WO93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993).

An antibody “which binds” a molecular target or an antigen of interestis one capable of binding that antigen with sufficient affinity suchthat the antibody is useful in targeting a cell expressing the antigen.

An antibody that “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide is onethat binds to that particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope. In such embodiments, the extent of binding of theantibody to these other polypeptides or polypeptide epitopes will beless than 10%, as determined by fluorescence activated cell sorting(FACS) analysis or radioimmunoprecipitation (RIA), relative to bindingto the target polypeptide or epitope.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)of mostly human sequences, which contain minimal sequence derived fromnon-human immunoglobulin. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which residues from ahypervariable region (also CDR) of the recipient are replaced byresidues from a hypervariable region of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, “humanized antibodies” as used hereinmay also comprise residues which are found neither in the recipientantibody nor the donor antibody. These modifications are made to furtherrefine and optimize antibody performance. The humanized antibodyoptimally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature, 321:522-525 (1986); Reichmannet al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992).

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and Bcell activation.

“Antibody-dependent cell-mediated cytotoxicity” or ADCC refers to a formof cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., natural killer (NK) cells,neutrophils and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are required for killing of the target cell by this mechanism.The primary cells for mediating ADCC, NK cells, express FcγRIII only,whereas monocytes express FcγRI, FcγRII and FcγRIII. Fc expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol. 9: 457-92 (1991). To assess ADCC activity ofa molecule of interest, an in vitro ACDD assay, such as that describedin U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and natural killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal., PNAS USA 95:652-656(1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors, FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see M. Daron, Annu.Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet,Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995).Other FcRs, including those to be identified in the future, areencompassed by the term “FcR” herein. The term also includes theneonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus. Guyer et al., J. Immunol. 117: 587 (1976)and Kim et al., J. Immunol. 24: 249 (1994).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils,with PBMCs and MNK cells being preferred. The effector cells may beisolated from a native source, e.g., blood.

“Complement dependent cytotoxicity” of “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202: 163 (1996), may be performed.

The terms “conjugate,” “conjugated,” and “conjugation” refer to any andall forms of covalent or non-covalent linkage, and include, withoutlimitation, direct genetic or chemical fusion, coupling through a linkeror a cross-linking agent, and non-covalent association, for exampleusing a leucine zipper. Antibody conjugates have another entity, such asa cytotoxic compound, drug, composition, compound, radioactive element,or detectable label, attached to an antibody or antibody fragment.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, non-human higher primates, domestic and farmanimals, and zoo, sports, or pet animals, such as dogs, horses, rabbits,cattle, pigs, hamsters, mice, cats, etc. Preferably, the mammal ishuman.

A “disorder” is any condition that would benefit from treatment with theprotein. This includes chronic and acute disorders or diseases includingthose pathological conditions which predispose the mammal to thedisorder in question.

A “therapeutically effective amount” is at least the minimumconcentration required to effect a measurable improvement or preventionof a particular disorder. Therapeutically effective amounts of knownproteins are well known in the art, while the effective amounts ofproteins hereinafter discovered may be determined by standard techniqueswhich are well within the skill of a skilled artisan, such as anordinary physician.

II. Modes for Carrying Out the Invention

A. Protein Preparation

In accordance with the present invention, the protein is produced byrecombinant DNA techniques, i.e., by culturing cells transformed ortransfected with a vector containing nucleic acid encoding the protein,as is well known in art.

Preparation of the protein by recombinant means may be accomplished bytransfecting or transforming suitable host cells with expression orcloning vectors and cultured in conventional nutrient media modified asappropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences. The cultureconditions, such as media, temperature, pH and the like, can be selectedby the skilled artisan without undue experimentation. In general,principles, protocols, and practical techniques for maximizing theproductivity of cell cultures can be found in Mammalian CellBiotechnology: A Practical Approach, M. Butler, Ed. (IRL Press, 1991)and Sambrook et al., Molecular Cloning: A Laboratory Manual, New York:Cold Spring Harbor Press. Methods of transfection are known to theordinarily skilled artisan, and include for example, CaPO₄ and CaCl₂)transfection, electroporation, microinjection, etc. Suitable techniquesare also described in Sambrook et al., supra. Additional transfectiontechniques are described in Shaw et al., Gene 23: 315 (1983); WO89/05859; Graham et al., Virology 52: 456-457 (1978) and U.S. Pat. No.4,399,216.

The nucleic acid encoding the desired protein may be inserted into areplicable vector for cloning or expression. Suitable vectors arepublicly available and may take the form of a plasmid, cosmid, viralparticle or phage. The appropriate nucleic acid sequence may be insertedinto the vector by a variety of procedures. In general, DNA is insertedinto an appropriate restriction endonuclease site(s) using techniquesknown in the art. Vector components generally include, but are notlimited to, one or more of a signal sequence, an origin of replication,one or more marker genes, and enhancer element, a promoter, and atranscription termination sequence. Construction of suitable vectorscontaining one or more of these components employs standard ligationtechniques which are known to the skilled artisan.

Forms of the protein may be recovered from culture medium or from hostcell lysates. If membrane-bound, it can be released from the membraneusing a suitable detergent or through enzymatic cleavage. Cells employedfor expression can also be disrupted by various physical or chemicalmeans, such as freeze-thaw cycling, sonication, mechanical disruption orcell lysing agents.

Purification of the protein may be effected by any suitable techniqueknown in the art, such as for example, fractionation on an ion-exchangecolumn, ethanol precipitation, reverse phase HPLC, chromatography onsilica or cation-exchange resin (e.g., DEAE), chromatofocusing,SDS-PAGE, ammonium sulfate precipitation, gel filtration using protein ASepharose columns (e.g., SEPHADEX® G-75) to remove contaminants such asIgG, and metal chelating columns to bind epitope-tagged forms.

B. Antibody Preparation

In certain embodiments of the invention, the protein of choice is anantibody. Techniques for the production of antibodies, includingpolyclonal, monoclonal, humanized, bispecific and heteroconjugateantibodies follow.

(i) Polyclonal Antibodies.

Polyclonal antibodies are generally raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor. Examples of adjuvants which may be employedinclude Freund's complete adjuvant and MPL-TDM adjuvant (monophosphorylLipid A, synthetic trehalose dicorynomycolate). The immunizationprotocol may be selected by one skilled in the art without undueexperimentation.

One month later the animals are boosted with ⅕ to 1/10 the originalamount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(ii) Monoclonal Antibodies.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986).

The immunizing agent will typically include the protein to beformulated. Generally either peripheral blood lymphocytes (“PBLs”) areused if cells of human origin are desired, or spleen cells or lymph nodecells are used if non-human mammalian sources are desired. Thelymphoctyes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell. Goding, Monoclonal antibodies: Principles and Practice, AcademicPress (1986), pp. 59-103. Immortalized cell lines are usuallytransformed mammalian cell, particularly myeloma cells of rodent, bovineand human origin. Usually, rat or mouse myeloma cell lines are employed.The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA. Human myeloma and mouse-human heteromyeloma cell lines alsohave been described for the production of human monoclonal antibodies(Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., MonoclonalAntibody Production Techniques and Applications, pp. 51-63 (MarcelDekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal.

The immunizing agent will typically include the epitope protein to whichthe antibody binds. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell. Goding, Monoclonal Antibodies: Principals and Practice, AcademicPress (1986), pp. 59-103.

Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myelome cell lines are employed. The hybridoma cells may becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Rockville, Md. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies. Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63.

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theprotein to be formulated. Preferably, the binding specificity ofmonoclonal antibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods.Goding, supra. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol.,5:256-262 (1993) and Plückthun, Immunol. Revs. 130:151-188 (1992).

In a further embodiment, antibodies can be isolated from antibody phagelibraries generated using the techniques described in McCafferty et al.,Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991)and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe theisolation of murine and human antibodies, respectively, using phagelibraries. Subsequent publications describe the production of highaffinity (nM range) human antibodies by chain shuffling (Marks et al.,Bio/Technology, 10:779-783 (1992)), as well as combinatorial infectionand in vivo recombination as a strategy for constructing very largephage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalmonoclonal antibody hybridoma techniques for isolation of monoclonalantibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

(iii) Humanized and Human Antibodies.

The antibodies subject to the formulation method may further comprisehumanized or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementarity determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domain,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Jones et al., Nature 321: 522-525 (1986); Riechmann etal., Nature 332: 323-329 (1988) and Presta, Curr. Opin. Struct. Biol. 2:593-596 (1992).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers,Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988), orthrough substituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody. Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies. Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J Immnol., 151:2623 (1993).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993). Human antibodies can also be derivedfrom phage-display libraries (Hoogenboom et al., J Mol. Biol., 227:381(1991); Marks et al., J. Mol. Biol., 222:581-597 (1991)).

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries. Hoogenboom and Winter, J.Mol. Biol. 227: 381 (1991); Marks et al., J. Mol. Biol. 222: 581 (1991).The techniques of Cole et al., and Boerner et al., are also availablefor the preparation of human monoclonal antibodies (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) andBoerner et al., J Immunol. 147(1): 86-95 (1991). Similarly, humanantibodies can be made by introducing human immunoglobulin loci intotransgenic animals, e.g., mice in which the endogenous immunoglobulingenes have been partially or completely inactivated. Upon challenge,human antibody production is observed, which closely resemble that seenin human in all respects, including gene rearrangement, assembly andantibody repertoire. This approach is described, for example, in U.S.Pat. Nos. 5,545,807; 5,545,806, 5,569,825, 5,625,126, 5,633,425,5,661,016 and in the following scientific publications: Marks et al.,Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859(1994); Morrison, Nature 368: 812-13 (1994), Fishwild et al., NatureBiotechnology 14: 845-51 (1996), Neuberger, Nature Biotechnology 14: 826(1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

(iv) Antibody Dependent Enzyme-Mediated Prodrug Therapy (ADEPT)

The antibodies of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g. a peptidyl chemotherapeutic agent, see WO 81/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such as way so as toconvert it into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, glycosidase, glucose oxidase, human lysozyme, humanglucuronidase, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases (e.g., carboxypeptidase G2and carboxypeptidase A) and cathepsins (such as cathepsins B and L),that are useful for converting peptide-containing prodrugs into freedrugs; D-alanylcarboxypeptidases, useful for converting prodrugs thatcontain D-amino acid substituents; carbohydrate-cleaving enzymes such asβ-galactosidase and neuraminidase useful for converting glycosylatedprodrugs into free drugs; β-lactamase useful for converting drugsderivatized with β-lactams into free drugs; and penicillin amidases,such as penicillin Vamidase or penicillin G amidase, useful forconverting drugs derivatized at their amine nitrogens with phenoxyacetylor phenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”can be used to convert the prodrugs of the invention into free activedrugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzymeconjugates can be prepared as described herein for delivery of theabzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the anti-IL-17or anti-LIF antibodies by techniques well known in the art such as theuse of the heterobifunctional cross-linking agents discussed above.Alternatively, fusion proteins comprising at least the antigen bindingregion of the antibody of the invention linked to at least afunctionally active portion of an enzyme of the invention can beconstructed using recombinant DNA techniques well known in the art (see,e.g. Neuberger et al., Nature 312: 604-608 (1984)).

(iv) Bispecific and Polyspecific Antibodies

Bispecific antibodies (BsAbs) are antibodies that have bindingspecificities for at least two different epitopes. Such antibodies canbe derived from full length antibodies or antibody fragments (e.g.F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities. Millstein et al.,Nature, 305:537-539 (1983). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 and in Traunecker et al., EIBOJ, 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions, and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies, see, for example,Suresh et al., Methods in Enzymology 121: 210 (1986).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g., tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechains(s) are created on the interface of the second antibody moleculeby replacing large amino acid side chains with smaller ones (e.g.,alanine or threonine). This provides a mechanism for increasing theyield of the heterodimer over other unwanted end-products such ashomodimers.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690, published Mar. 3,1994. For further details of generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology, 121:210 (1986).

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373). Heteroconjugate antibodies may be made usingany convenient cross-linking methods. Suitable cross-linking agents arewell known in the art, and are disclosed in U.S. Pat. No. 4,676,980,along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. The following techniques canalso be used for the production of bivalent antibody fragments which arenot necessarily bispecific. For example, Fab′ fragments recovered fromE. coli can be chemically coupled in vitro to form bivalent antibodies.See, Shalaby et al., J Exp. Med., 175:217-225 (1992).

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229: 81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)₂ fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-TNB derivative to form the bispecificantibody. The bispecific antibodies produced can be used as agents forthe selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J Exp. Med. 175:217-225 (1992) describes the production of fully humanized bispecificantibody F(ab′)₂ molecules. Each Fab′ fragment was separately secretedfrom E. coli and subjected to directed chemical coupling in vitro toform the bispecific antibody. The bispecific antibody thus formed wasable to bind to cells overexpressing the ErbB2 receptor and normal humanT cells, as well as trigger the lytic activity of human cytotoxiclymphocytes against human breast tumor targets.

Various techniques for making and isolating bivalent antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bivalent heterodimers have been produced using leucine zippers.Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucinezipper peptides from the Fos and Jun proteins were linked to the Fab′portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. The “diabody” technologydescribed by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448(1993) has provided an alternative mechanism for makingbispecific/bivalent antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific/bivalentantibody fragments by the use of single-chain Fv (sFv) dimers has alsobeen reported. See Gruber et al., J Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J Immunol. 147: 60(1991).

Exemplary bispecific antibodies may bind to two different epitopes on agiven molecule. Alternatively, an anti-protein arm may be combined withan arm which binds to a triggering molecule on a leukocyte such as aT-cell receptor molecule (e.g., CD2, CD3, CD28 or B7), or Fc receptorsfor IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16)so as to focus cellular defense mechanisms to the cell expressing theparticular protein. Bispecific antibiotics may also be used to localizecytotoxic agents to cells which express a particular protein. Suchantibodies possess a protein-binding arm and an arm which binds acytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTAor TETA. Another bispecific antibody of interest binds the protein ofinterest and further binds tissue factor (TF).

(v) Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells, U.S. Pat. No. 4,676,980,and for treatment of HIV infection. WO 91/00360, WO 92/200373 and EP03089. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

C. Purification of the Proteins, Including Antibodies

When the target polypeptide is expressed in a recombinant cell otherthan one of human origin, the target polypeptide is completely free ofproteins or polypeptides of human origin. However, it is necessary topurify the target polypeptide from recombinant cell proteins orpolypeptides to obtain preparations that are substantially homogeneousas to the target polypeptide. As a first step, the culture medium orlysate is typically centrifuged to remove particulate cell debris. Themembrane and soluble protein fractions are then separated. The targetpolypeptide may then be purified from the soluble protein fraction andfrom the membrane fraction of the culture lysate, depending on whetherthe target polypeptide is membrane bound. The following procedures areexemplary of suitable purification procedures: fractionation onimmunoaffinity or ion-exchange columns; ethanol precipitation; reversephase HPLC; chromatography on silica or on a cation exchange resin suchas DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; and protein A Sepharosecolumns to remove contaminants such as IgG.

Most companies currently producing monoclonal antibodies (MAbs) use athree-column platform approach comprising Protein A affinitychromatography for product capture, followed by anion exchangechromatography in flow-through mode to extract negatively chargedcontaminants such as host cell protein (HCP), endotoxins, host DNA, andleached Protein A, and then cation exchange chromatography orhydrophobic interaction chromatography (HIC) in retention mode to removepositively charged contaminant species including residual HCP andproduct aggregates.

Those viruses that may be present in protein solutions are larger thanthe proteins themselves. It is thus presumed that viruses can be removedfrom proteins in accordance with size, by filtration.

Virus filtration can remove larger, e.g., retroviruses (80-100 nmdiameter), typically using high throughput membranes with nominal poresize of about 60 nm. Since high throughput membranes with nominal poresize of 20 nm are also commercially available, it is possible to removesmaller viruses by filtration, such as, for example, parvoviruses (18-26nm diameter), while allowing passage of proteins that are as large as160 kD (˜8 nm), e.g., monoclonal antibodies. The present invention isprimarily intended for resolving issues typically associated with thefiltration of such smaller viruses, using viral removal filters ofsmaller pore size.

Typically, a virus filtration step can be implemented at any one ofseveral points in a given downstream process. For example, in a typicalmonoclonal antibody purification process, virus filtration may takeplace following a low pH viral inactivation step, or following anintermediate column chromatography step, or after a final columnchromatography step.

According to the invention, virus filtration unit operation could becarried out at any stage in the downstream process. Virus filtrationduring downstream processing of monoclonal antibody is typicallyperformed after an affinity chromatography step (capture step) and anion-exchange purification step (polishing step).

The experimental setup used in the experiments disclosed herein isillustrated in FIG. 1. It is emphasized, however, that the invention isnot so limited. Other arrangements, well known in the art, are alsosuitable and can be used in the methods of the present invention.

In tangential flow virus filtration, the protein solution is usuallypumped around at a constant rate of flow on the retention side. Thedifferential pressure generated across the virus removal filter, allowsprotein solution to permeate through the filter while the viruses areretained on the retentate side.

In the case of so called “normal-flow” or “dead-end” virus filtration,the same virus filter as that used in tangential virus filtration can beused, although the peripheral equipment and operating procedures aremuch simpler and less expensive than in the case of tangential flowvirus filtration. Thus, in principle, “normal-flow” filtration involvesplacing the macromolecule-containing solution in a pressure vessel priorto filtration and pressing the solution through the virus removal filterwith the aid of a pressure source, suitably nitrogen (gas) or air.Alternatively, a pump could be used on the retentate side to filter theliquid through the virus removal filter at a pre-determined flow rate.

The degree of fineness of filters generally, is normally expressed aspore size or the approximate molecular weight (relative molecular mass)at which the molecules are stopped by the filter, the so called cut-off.

Virus filters are known in the art and are supplied by Millipore fromMassachusetts, USA and Asahi Chemical Industry Co., Ltd. from Japan,among others. Suitable parvovirus retentive filters include VIRESOLVE®Pro (Millipore Corp., Billerica, Mass.) VIRESOLVE® Pro membrane has anasymmetric dual layer structure and is made from polyethersulfone (PES).The membrane structure is designed to retain viruses greater than 20 nmin size while allowing proteins of molecular weight less than 180 kDa topermeate through the membrane. Other filters suitable for the removal ofsmall viruses, including parvoviruses, from protein solutions includeNOVASIP™ DV20 and DV50 Virus Removal Filter Capsules (Pall Corp., EastHills, N.Y.), VIROSART® CPV, Planova 20 N (Asahi Kasei) and BioEX (AsahiKasei). The NOVASIP™ DV20 grade capsule filter utilizes an ULTIPOR®VF-grade DV20 grade pleated membrane cartridge to remove parvovirusesand other viruses as small as 20 nm from protein solutions up to 5-10liters. The NOVASIP™ DV50 grade capsule filter incorporates an ULTIPOR®VF DV50 grade ULTIPLEAT® membrane cartridge for removal of viruses 40-50nm and larger. NOVASIP® ULTIPOR® VF capsule filters are suppliednon-sterile and can also be Gamma-irradiated. VIROSART® CPV utilizesdouble-layer polyethersulfone asymmetric membrane and retains more than4 log of parvoviruses and 6 log of retroviruses.

Prefiltration of the feed solution can have a dramatic impact on filterperformance. Prefiltration typically is targeted to remove impuritiesand contaminants that might lead to fouling of virus filters, such asprotein aggregates, DNA and other trace materials.

According to the present invention, a striking enhancement of theefficacy of virus filters can be achieved by a prefiltration stepincluding the use of both cation exchange and endotoxin removal media.In this context, the term “medium” or “media” is used to cover any meansfor performing the cation exchange and endotoxin removal steps,respectively. Thus, the term “cation exchange medium” specificallyincludes, without limitation, cation exchange resins, matrices,absorbers, and the like. The term “endotoxin removal medium” includes,without limitation, any positively charged membrane surface, including,for example, chromatographic endotoxin removal media, endotoxin affinityremoval media, and the like.

Cation exchange media suitable for use in the prefiltration step of thepresent invention include, without limitation, MUSTANG® S, SARTOBIND® S,VIRESOLVE® Shield, SPFF, SPXL, CAPTO® S, POROS® 50 HS, FRACTOGEL® S,HYPERCEL® D etc., which are commercially available.

Endotoxin removal media suitable for use in the prefiltration step ofthe present invention include, without limitation, MUSTANG® E, MUSTANG®Q, SARTOBIND® Q, CHROMASORB®, POSSIDYNE®, CAPTO® Q, QSFF, POROS® Q,FRACTOGEL® Q etc., which are commercially available.

The pre-filtration step can be performed, for example, by taking the inprocess chromatography pool and processing the pool over a filtrationtrain that comprises the endotoxin removal and cation exchange media andparvovirus filter. The endotoxin removal and cation exchange media actas pre-filtration steps and the capacity of parvovirus filter isindependent of the sequence of two steps in the filtration train. Thefiltration train can work continuously as a single step or it can beoperated as different unit operations. For example, in one embodiment,the chromatography pool is first processed over endotoxin removal media,the collected pool is then processed over cation exchange media and thesubsequent pool is filtered with parvovirus filter. As mentioned above,the order of applying the cation exchange media and endotoxin removalmedia in the process sequence does not impact parvovirus filtrationcapacity. The process can be operated over a wide pH range, such as, forexample, in the pH range of 4-10, with optimal filter capacity beingdependent on the target impurity profile and product attributes.Similarly, protein concentrations can vary over a wide range, such as,for example, 1-40 g/L, and does not limit the mass throughput ofparvovirus filters.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All citations throughout the disclosure arehereby expressly incorporated by reference.

EXAMPLE

Materials and Methods

1. Protein Solution

Since virus filtration during downstream processing of monoclonalantibody is performed after the affinity chromatography (capture step)and an ion-exchange step (polishing step), all filtration experimentswere performed with commercially relevant in process ion exchange(cation or anion-exchange) chromatography pools. The mAb concentrationand pool conductivity for cation exchange and anion exchange pools wererespectively 10 mg/ml and 10 mS/cm and 8 mg/ml and 4 ms/cm. Filtrationexperiments were performed either with fresh feedstock (used within 24hours of production) or with feedstock that was frozen at −70° C. afterproduction and was thawed at 4-8° C. prior to use. No significantdifference was seen in results obtained with fresh or freeze-thawedfeedstock. Protein concentration was determined using a UV-visspectrophotometer (NanoDrop ND-1000, NanoDrop Technologies, Wilmington,Del.) with absorbance measured at 280 nm.

2. Membranes

Filtration experiments were performed with VIRESOLVE® Pro (MilliporeCorp., Billerica, Mass.) parvovirus retentive filter. VIRESOLVE® Promembrane has an asymmetric dual layer structure and is made frompolyethersulfone (PES). The membrane structure is designed to retainviruses greater than 20 nm in size while allowing proteins of molecularweight less than 180 kDa to permeate through the membrane. Prefilters toVIRESOLVE® Pro evaluated in this study included VIRESOLVE® Optiscale 40depth filter (Millipore Corp., Billerica, Mass.), FLUORODYNE® EX Mini0.2 μm sterile filter (Pall Corp., East Hills, N.Y.) and the membraneadsorbers from MUSTANG® family (Pall Corp., East Hills, N.Y.). Themembrane adsorbers were procured from the vendor in fully encapsulatedACRODISC® units. Table 1 summarizes the key properties (functionalgroup, bed volume, pore size etc.) of all the pre-filters used in thisstudy.

TABLE 1 Key Properties of Prefilters Functional Bed Volume/ PorePrefilter Utility Base Matrix Group Surface Area Size VIRESOLVE ® DepthFilter Diatomaceous — — — Earth FLURODYNE ® EX Sterile Filter Polyethersulfone — 3.8 cm2 0.2 μm MUSTANG ® S Strong Cation Polyether sulfoneSulfonic Acid 0.18 ml 0.8 μm Exchanger MUSTANG ® Q Strong AnionPolyether sulfone Quaternary 0.18 ml 0.8 μm Exchanger Amine MUSTANG ® EEndotoxin Polyether sulfone Polyethylene 0.12 ml 0.2 μm Removal Imine

3. Experimental Setup

Filtration experiments were performed with a custom-built apparatusshown in FIG. 1. The load material, i.e., in process mAb pool, wasplaced in the load reservoir and was filtered through a filtration trainconsisting of different combinations of pre-filters and commerciallyavailable parvovirus filters. In all filtration experiments, theconstant filtration flow rate (P_(max)) method was used. Pressuretransducers were placed upstream of each filter and were coupled to aMillidaq or a Netdaq system to record differential pressure data as afunction of time or mass throughput. Filtrate from the parvovirus filterwas collected in a reservoir, which was kept on a load cell to recordmass throughput as a function of time.

Results and Discussion

Downstream purification of mAbs expressed in mammalian cell culturestypically utilize centrifugation and depth filtration as a first step toremove cells and cell debris, followed by affinity chromatography formAb capture and removal of host cell proteins (HCP), followed by cationexchange chromatography, virus filtration, and anion exchangechromatography for further removal of impurities such as aggregates,viruses, leached protein A and HCP's. Majority of the experiments inthis study were performed with cation exchange pool with cation exchangechromatography being the second chromatography step.

FIG. 2 shows the experimental data for differential pressure acrossViresolve Pro at a constant flux of 200 L/m²/hr with a therapeutic mAbfeed stream with different prefilters. X-axis represents the mass of mAbloaded per square meter of virus filter. Y-axis represents thedifferential pressure across the virus filter as a function of massthroughput. The data clearly indicates that the depth filter providesseveral orders of magnitude increase in virus filtration capacitycompared to sterile filter. Similar observations were made by Bolton etal. (Bolton et al. Appl. Biochem. 43:55-63, 2006) when evaluating theeffect of VIRESOLVE Prefilter™—a depth filter media—as a pre-filter toNFP parvovirus retentive filter (Millipore Corp.) with a polyclonal IgGsolution. The authors attributed the increase in capacity to theselective adsorption of foulant—denatured protein—due to hydrophobicinteractions.

Although depth filters have traditionally been used successfully forclarification of cell culture fluid, there are quite a few limitationsthat deserve extra consideration when used downstream of capture steps,e.g., as a prefilter to parvovirus retentive filter.

-   -   (a) Depth filters are not base stable which prevents the        sanitization of process train after installation, resulting in        open processing and potential for bioburden growth.    -   (b) Composition of depth filters includes diatomaceous earth as        a key component, which is typically food grade and presents        quality concerns.    -   (c) The diatomaceous earth is generally sourced from        nature—lacking a well defined chemical process—and can thus can        have lot to lot variations.    -   (d) Depth filters also tend to leach metals, beta-glycans and        other impurities, the clearance of which needs to be        demonstrated and validated with downstream operations.

These limitations put extra burden on process development as the unitoperations downstream of depth filter would have to be designed toprovide adequate clearance of leachables. However, even if the requisiteof leachable clearance was met, there are reasons to be concerned that aparticular lot of depth filter may have significantly higher leachablesthan what the process is capable of clearing as the key components aresourced from nature, that is, they lack a well defined chemicalsynthesis process.

There has thus been a significant interest in development of pre-filtersthat do not present these limitations. As mentioned above, Brown et al.(Brown et al. IBC's 20^(th) Antibody Development and Production, SanDiego, Calif., 2008) recently showed that Mustang S, a strong negativelycharged ion-exchanger, when used as a prefilter could increase thecapacity of parvovirus retentive filter by several fold. Experimentswere thus conducted to evaluate the effect of different prefiltrationmedia to Viresolve® Pro. The experimental data at pH 5.0 and 6.5 isshown in FIGS. 3 (a) & (b). The data shows that while cation exchangemedia shows slight benefit over endotoxin removal adsorber at pH 5.0,the benefit disappeared at pH 6.5. While the overall capacities withboth media were higher than those with sterile filter (FIG. 2); theywere nonetheless significantly short of the capacity required tosuccessfully conduct the unit operation at manufacturing scale.

Based on the hypothesis that both cation exchange and endotoxin removalmedia could be removing two different foulants; both of which may leadto filter fouling, an experiment was designed with a novel prefiltrationtrain that included both cation exchange and endotoxin removal media.Experimental results are shown in FIGS. 4 (a) and (b) at pH 5.0 and pH6.5 respectively. The data clearly indicate that the combination of twomedia is significantly better than each of the media by itself. Forexample, at pH 5.0, the combination of cation exchange and endotoxinremoval media provide greater than an order of magnitude improvement incapacity at 20 psi differential pressure. While similar trend was alsoseen at pH 6.5, the overall capacity was lower than that obtained at pH5.0. It could be due to more robust removal of impurities at lower pH.

Experimental results with MAb2 are shown in FIG. 5. Consistent with datain FIG. 4, the novel prefiltration train containing both endotoxinremoval media and cation exchange media increased the capacitysubstantially, suggesting that endotoxin removal media and cationexchange media work synergistically and remove two different classes offoulants.

CONCLUSIONS

Majority of the previous work has focused on the use of depth filters orcation exchange membrane adsorbers as prefilters to increase thecapacity of parvovirus retentive filters. While depth filters provide arobust mechanism for increasing virus filtration capacity, limitationsassociated with them such as leachables limit their application to aspecific stage in the downstream purification sequence. Whilecation-exchange membrane adsorbers may increase the parvovirus filtercapacity for some monoclonal antibody (mAb) feedstreams, they may not beuniversally applicable as seen with the data in this study, suggestingthat there may be multiple foulants present, which need to be addressedto further improve performance of parvovirus removal filters.

The present invention, as demonstrated by the above experimentalresults, highlights two aspects—(1) Endotoxin removal media by itselfcan effectively increase the capacity of parvovirus filters when usedfor prefiltration and (2) coupling of endotoxin removal and cationexchange media in the prefiltration train can provide several-foldincrease in parvovirus filtration capacity, lowering raw material costsand facilitating successful operation of virus filtration atmanufacturing scale.

1. A method of improving the filtration capacity of a virus filterduring protein purification, comprising subjecting a compositioncomprising a protein to be purified to a cation exchange step and anendotoxin removal step, simultaneously or in either order, prior topassing through said virus filter.
 2. The method of claim 1 wherein thepore size of the virus filter is between about 15 and about 100 nmdiameter.
 3. The method of claim 2 wherein the pore size of the virusfilter is between about 15 and about 30 nm diameter.
 4. The method ofclaim 3 wherein the pore size of the virus filter is about 20 nm.
 5. Themethod of claim 3 wherein the virus to be removed is a parvovirus. 6.The method of claim 5 wherein the diameter of the parvovirus is betweenabout 18 and about 26 nm.
 7. The method of claim 1 wherein the proteinis an antibody or an antibody fragment.
 8. The method of claim 7 whereinthe protein is a recombinant antibody or antibody fragment.
 9. Themethod of claim 8 wherein the recombinant antibody or antibody fragmentis produced in a mammalian host cell.
 10. The method of claim 9 whereinthe mammalian host cell is a Chinese Hamster Ovary (CHO) cell.
 11. Themethod of claim 1 wherein the composition comprising the protein to bepurified is first subjected to the cation exchange step followed by theendotoxin removal step, prior to virus filtration.
 12. The method ofclaim 1 wherein the composition comprising the protein to be purified isfirst subjected to the endotoxin removal step followed by the cationexchange step, prior to virus filtration.
 13. The method of claim 1wherein the composition comprising the protein to be purified issubjected to the endotoxin removal step and the cation exchange stepsimultaneously, prior to virus filtration.
 14. The method of claim 11wherein said endotoxin removal step is directly followed by virusfiltration.
 15. The method of claim 12 wherein said cation exchange stepis directly followed by virus filtration.
 16. The method of claim 13wherein said simultaneous endotoxin removal and cation exchange step aredirectly followed by virus filtration.
 17. The method of claim 1 whereinvirus filtration is performed at a pH between about 4 and about
 10. 18.The method of claim 1 wherein the protein concentration in saidcomposition is about 1-40 g/L.
 19. The method claim 8 wherein saidantibody is to one or more antigens selected from the group consistingof HER1 (EGFR), HER2, HER3, HER4, VEGF, CD20, CD22, CD11a, CD11b, CD11c,CD18, an ICAM, VLA-4, VCAM, IL-17A and/or F, IgE, DR5, CD40,Apo2L/TRAIL, EGFL7, NRP1, mitogen activated protein kinase (MAPK), andFactor D.
 20. The method of claim 8 wherein the antibody is selectedfrom the group consisting of anti-estrogen receptor antibody,anti-progesterone receptor antibody, anti-p53 antibody, anti-cathepsin Dantibody, anti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125antibody, anti-CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB-2antibody, anti-P-glycoprotein antibody, anti-CEA antibody,anti-retinoblastoma protein antibody, anti-ras oncoprotein antibody,anti-Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody,anti-CD8 antibody, anti-CD9/p24 antibody, anti-CD10 antibody, anti-CD11cantibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody,anti-CD19 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody,anti-CD38 antibody, anti-CD41 antibody, anti-LCA/CD45 antibody,anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39 antibody,anti-CD100 antibody, anti-CD95/Fas antibody, anti-CD99 antibody,anti-CD106 antibody, anti-ubiquitin antibody, anti-CD71 antibody,anti-c-myc antibody, anti-cytokeratins antibody, anti-vimentinsantibody, anti-HPV proteins antibody, anti-kappa light chains antibody,anti-lambda light chains antibody, anti-melanosomes antibody,anti-prostate specific antigen antibody, anti-S-100 antibody, anti-tauantigen antibody, anti-fibrin antibody, anti-keratins antibody andanti-Tn-antigen antibody.